This invention relates to a method of potentiating cell damage by administering an agent that retards the rate of movement of a target cell through some portion of the cell-division cycle and administering a cytotoxic agent that acts within a portion of the cell-division cycle through which movement has been slowed. The method of the invention can be used in chemotherapy as well as in other medical and non-medical applications. In a specific embodiment, deoxythymidine (dThd) is the agent retarding the rate of movement of a target cell through a portion of the cell-division cycle and staurosporine is the cytotoxic agent. The invention also relates to a method of using a microculture indicator system (MIS) and auxiliary data analysis procedures to determine the degree of interaction between agents. In a specific embodiment, data are collected reflecting the effect on cell growth of two or more agents arrayed in serial bivariate dilutions, and a database is caused to process the data in a spreadsheet according to predetermined relationships with reference measurements of cell growth and to present, in graphical or tabular form, the spectrum of interaction of the agents with respect to reference measurements.
The use of drugs or other agents for destroying or inflicting permanent damage on living cells serves a number of valuable and legitimate objectives. A major clinical use is for the ablation of malignant tumors or other abnormal tissue growths. V T DeVita, Jr., IN: Cancer, Principles and Practice of Oncology, 4th ed., pp. 276-292, JB Lippincott Co. Philadelphia (1994).
Other valuable clinical uses have included (1) medical control of abnormal immunologic reactions, K Wilson et al., Rheumatol. 21:1674-7 (1994); C M Neuwelt et al., Am. J. Med. 98:32-41; (1995); (2) exfoliative dermatological disease, G D Weinstein et al., J. Am. Acad. Dermatol. 28:454-9 (1993), R J Van Dooren-Greebe et al., Br. J. Dermatol. 130:204-10 (1994); (3) killing of cells infected by viruses, viral replicative elements, or prions, P Calabresi et al., Section XIIxe2x80x94Chemotherapy of neoplastic diseases IN: Goodman and Gilman""s The Pharmacologic Basis of Therapeutics, 8th ed., 1202-1263 (Pergammon Press, New York 1990); S Chou et al., Antiviral chemotherapy, Chapter 17 IN: Virology, pp. 323-348, ed. B N Fields et al., Raven Press, New York (1985); (4) therapies for systemic or topical elimination of infective agents including bacteria, mycobacteria, mycoplasma, rickettsia, fungi, yeast, or parasitic organisms, H P Willett, The action of chemotherapeutic agents, Chapter 10, IN: Sinsser Microbiology, 17th ed., pp. 234-277, ed. Joklik et al., Appleton-Century-Crofts, NY (1980); V Lorian, Antibiotics in Laboratory Medicine, 3d ed., Williams and Wilkins, Baltimore (1991); S Stemberg, Science 266:1632-1634 (1994), (5) and fertility control. Nonclinical uses of agents capable of inflicting permanent damage on living cells occur in agriculture, horticulture, or public health, e.g., application of specific pesticides or herbicides.
A vast array of physical, chemical, or biological agents are hazardous to living cells and can inflict damage upon biological systems such as tissues or organs. In many cases, however, the damage is not specifically targeted to events related to the cell-division cycle.
In other cases, cell damage may be initiated in direct relation to the hierarchy of the cell-division cycle. A cytotoxic agent that acts during some portion of the cell-division cycle, causing biologically significant or irreversible damage to a proliferating cell, may serve as a xe2x80x9ctargeted cytotoxic insultxe2x80x9d or xe2x80x9cTCIxe2x80x9d, as defined herein. The portion of the cell-division cycle during which a given TCI initiates a relevant action is its xe2x80x9ctarget interval.xe2x80x9d
Known agents that can act as TCIs are diverse and include natural substances, products of microbial or other cellular origins, synthetic or semi-synthetic organic or inorganic chemical compounds, or simple inorganic reagents. Other factors that can act as a TCI are also known and may include deprivation of nutrients essential to cell growth or sustenance as well as changes in the physicochemical environment. Examples of the latter include temperature changes and exposure of the cells to radiant or particulate energies, vibrational waves, or various other physical forces.
Cytotoxic effects of a TCI may not be immediate, so that cell damage initiated in one phase of the cell-division cycle may not become manifest until a later phase or a subsequent cell cycle. As just one example, in cisplatin treatment, permanently injured progeny cells may be sterile or exhibit a reduced capacity to proliferate or survive. M Sorenson, J Natl. Cancer Inst. 82:749-55 (1990). Thus, an understanding of the cell-division cycle hierarchy becomes useful to further understanding of agents that can act as TCIS.
All growing cells must duplicate their genomic DNA and pass identical copies of this genetic information to their progeny. In order to accomplish this task, proliferating somatic (non-reproductive) and germ (reproductive) cells of all living organisms undergo repetitive cell-division cycles (hereinafter xe2x80x9ccell cyclexe2x80x9d or xe2x80x9cCCxe2x80x9d). Each completed cell-division cycle-results in the duplication of the cell""s genetic information and the division of the parent cell into two daughter cells, with an equal division of the parental cell DNA.
The biochemical and biomolecular processes that comprise the cell cycle include, among other things, enzyme-dependent DNA replication, enzyme-dependent phosphorylation, signal cascades, association and dissociation of transcriptional activating molecular complexes, and formation and dissociation of macromolecular assemblies of cytostructural elements including cytomembranes and the cytoskeleton.
A. Cell Cycle Hierarchy
The processes characterizing the cell cycle form a regulated hierarchy and advance in a strict order dependence under the control of a cell cycle xe2x80x9cenginexe2x80x9d or xe2x80x9ccontrol system.xe2x80x9d The control system functions as a biomolecular xe2x80x9cclockxe2x80x9d or xe2x80x9coscillatorxe2x80x9d and includes critical controls at xe2x80x9ccheckpoints.xe2x80x9d L N Edmunds, Jr., Ann. NY Acad. Sci. 719:77-96 (1994); I A Carre et al., J Cell Sci. 104:1163-73 (1993); B G Gabrielli et al., J. Biol Chem 267:1969-75 (1992); A Goldbeter, Proc. Natl. Acad. Sci. (USA) 88:9107-11 (1991); Murray A W and Kirschner M W, Science 246:614-621 (1989).
In the normal cell cycle hierarchy, DNA replication is followed by mitosis and cytokinesis. See generally A W Murray, Nature 359:599-604 (1992); B Alberts et al., The cell-division cycle, IN: Molecular Biology of the Cell, 3d edition, Garland Publishing Inc., New York (1994); B A Edgar et al., Genes Dev 8:440-52 (1994). A series of molecular processes, each process functioning in an appropriate order during the cell cycle, moves the cell in the direction of cell division with a downstream momentum. In this context, the term xe2x80x9cdownstreamxe2x80x9d refers to events that occupy a xe2x80x9csubordinate positionxe2x80x9d in the cell cycle hierarchy as defined by Alberts, supra. Order dependence in the cell cycle hierarchy ensures that DNA replication proceeds with maximal fidelity. See L H Hartwell et al., Science 246:629-634 (1989); P M O""Connor et al., Semin. Cancer Biol. 3:409-416 (1992).
The hierarchy of the eukaryotic cell cycle relates to four conserved functional landmarks (FIG. 1): S phase, in which nucleotides are synthesized and DNA is semi-conservatively replicated in double-stranded helixes of polynucleotides; G2 phase, which follows completion of DNA synthesis and during which DNA associates with nucleoproteins; M phase, in which nuclear filaments condense as chromosomes and chromosomes segregate for mitosis; and G1 phase, during which cells prepare for renewed division by replacement of depleted products and repair of any lesions in DNA. See Alberts, supra. Cells entering S phase normally are committed to completion of G2 phase, M phase, and cytokinesis.
B. Cell Cycle Checkpoints
Transitions between phases are the major checkpoints in the cell cycle. In normal cells, they are tightly regulated by a decision point in G1 (START) and checkpoint controls associated with the boundaries between G1 and S (G1/S) and G2 and M (G2/M). See K A Heichman et al., Cell 79:557-562 (1994); P Nurse, Cell 79:547-550 (1994); A W Murray, supra; A W Murray et al., Sci. Am. 264:56-63 (1991); Hartwell, supra. Controlled interactions of specific proteins such as cyclins, cyclin-dependent kinases (cdk or cdc), and a series of accessory proteins (including p16, p21, p27, p45 or p53), which regulate cdk or cdc cyclin complexes, regulate successive phases of the cell cycle. T Hunter et al., Cell 79:573-582 (1994); Heichman, supra.; Nurse, a.; R W King et al., Cell 79:563-571 (1994); L H Tsai et al., Oncogene 8: 1593-602 (1993); M Doree et al., FASEB J. 8:1114-1121 (1991). Moreover, function of p53 and phosphorylation of the Rb tumor suppressor gene product (pRb) are also associated with the G1/S transition; V Karantza et al., Mol. Cell Biol. 13:6640-52 (1993); M E Ewen et al., Cell 73:487-97 (1993); S J Kuerbitz et al., Proc. Natl. Acad. Sci. USA 89:7491-95 (1992); M B Kastan Cell, 71:587-597 (1992).
C. Cell Cycle Kinetics
In a population of cells, the mean duration of each cell cycle phase is proportional to the probability of finding a cell within a given phase. If it is assumed that no loss, quiescence, or differentiation of progeny cells will occur during the continuous proliferation of a cell population, then for any given population, i.e., cohort the mean duration of a single cell cycle will equal the time to double; i.e., the time required for the starting cell population to double, i.e., its generation time. See, L A Perez et al., Cancer Res. 55:392-398 (1995). This concept can be expressed mathematically. Thus, TDBL is defined as the time required for the present population (NP) divided by the original population (N0) to double (N/N0=2).
The TDBL provides a yardstick for determining the xe2x80x9cfractional durationxe2x80x9d of major phases in the cell cycle phases, i.e., the time required for a fraction of the cell population to complete G1 (TG1), S (TS), or G2 and M (TG2 and M). Assuming continuous proliferation of an ideal cohort without any loss, quiescence, or differentiation of progeny, the fractional duration of each phase of the cell cycle is directly proportional to the fraction of the cell population (F) that is cycling through that phase at any moment in time, i.e., the FG1, FS or FG2 and M; for example, TS, can be calculated as TS=FSxc3x97TDBL. See Alberts, supra, and Perez, supra. Practically, this is an important equation since TDBL can be determined from serial cell counts, or flow cytometry, A C Begg et al., Cytometry 6:620-626 (1985), while FS, FG2+M or FG1/G0 can be measured in DNA histograms obtained from flow cytometry.
In standard flow cytometry, nuclei are stained with propidium iodide, a dye which intercalates into the minor groove of DNA. N M Shapiro, Practical Flow Cytometry, Alan R Liss, NY (1988). Histograms of dye absorbance discriminate the fractions of cells with different amounts of DNA/nucleus. Thus cells in the process of synthesizing DNA (S phase), or with a complete duplication of DNA (G2 phase and M phase), are distinguished from cells that have not begun to replicate their DNA (G1 or G0). Flow cytometric DNA histograms are reproducible and accurate under most testing conditions. See Perez, supra; J Pierrez et al., Acta Biotheor. 40: 131-7 (1992). DNA synthesis is also measured by BudR incorporation (Begg, supra).
When changes in physiological stimuli or ambient growth conditions slow down the growth of a cell population, the fraction of cells found in G1 phase (the preparation phase) typically increases at the expense of cells in S phase or in the growth fraction, i.e., G2 and M phases. Such changes or abnormal conditions may include hormonal, nutritional, or environmental changes. If abnormal conditions prevail, then cells in G1 phase may retire temporarily from the cell cycle to become xe2x80x9cquiescentxe2x80x9d or non-active. Quiescent cells are commonly designated to be in G0 phase. R Baserga, Cell Division, Molecular Biology, IN: Encyclopedia of Human Biology 2:253-266 (1991); A B Pardee, Science 246:609-613 (1989).
The process of differentiation, or specialization of cells, is also associated with a retirement of cells to G0. In terminal differentiation, the transition out of the cell cycle, i.e., into G0, becomes irreversible. Examples of terminally differentiated cells are adult neurons, keratinized epithelia, and voluntary muscle cells.
D. Apoptosis
Apoptosis is referred to as a process of xe2x80x9cprogrammed cell death.xe2x80x9d During normal somatic development, cell populations in specific organs or tissues may be programmed for death as part of the developmental progression of tissue remodeling or obsolescence. See J J Cohen, Avd. Immunol 50:55-85 (1991); M Baringa, Science 259:762-3 (1993). Apoptosis is internally triggered by biochemical or biomolecular mechanisms intrinsic to the cell cycle, resulting in an activation of endogenous endonucleases (enzymes that degrade DNA), leading to DNA strand breaks between nucleosomes and degradation of the genomic DNA by fragmentation. A H Wyllie, Nature 284:555-6 (1980). Apoptosis in mature tissues occurs in normal processes such as inflammation or rejuvenation. M Schmied et al., Am J Pathol 143:446-52 (1993); Abnormal clonal proliferations in immunologic diseases or malignancies may be related to a failure of normal apoptosis. J Marx, Science 259:760-1 (1993).
The relationship of apoptosis and/or cell damage to the cell cycle, including checkpoint controls, during cancer chemotherapy is a subject of interest to oncologists and molecular biologists. See T Shimizu et al., Cancer Res. 55:228-231 (1995); O""Connor, supra. (1992). The expression of p53 in damaged cells is one factor in determining the course of divergent biochemical pathways, which can lead to either DNA repair or apoptosis. E Yonish-Rouach et al., Mol Cell Biol 13:1415-23 (1993); D E Fisher, Cell 78:539-542 (1994).
Conflicting signals in the cell-division cycle may underlie the diversion of cell activities from proliferation to apoptosis. Fisher, supra. Cells entering or traversing the cell cycle transition boundaries or in the process of DNA replication or repair are most susceptible to apoptosis. In cells treated with a TCI, or in neoplastic cells, checkpoint controls such as cyclin-dependent kinases may be deregulated. This deregulation can release DNA replication or cell division events from START and homeostatic order dependence, intensifying cell damage. Id.
Current models of cancer chemotherapy are based largely upon two central dogmas. First, any mass of tumor cells which is clinically detectable must include a significant number of cells which will exhibit some biologically significant level of resistance to any single chemotherapeutic agent. J H Goldie et al., Cancer Treat Rep. 53:1727-1733 (1979). Second, according to the accepted Gompertzian model, tumor cell killing relates to the fraction of cells in active growth. L A Norton, Cancer Res. 48:7067-71 (1988).
In chemotherapy for malignancy, treatments with TCI have involved a number clinical considerations: they may be used in the primary effort to control cancer (induction chemotherapy), or as an adjunct to surgery or radiotherapy (adjuvant chemotherapy). DeVita, supra (1994). Local treatments have included infusion of TCI into body cavities to control the spread of malignancies such as breast or ovarian cancers. Id.
A. Single Agent Chemotherapy
In single agent induction, the usual objective is to administer the highest safe and tolerated dose to achieve maximal cancer cell killing or growth arrest. However, due to a cancer patient""s decreased ability to mount a cell mediated immune response against malignant cells, single agent chemotherapies rarely prove sufficient to control cancer in the human body. Neoplastic (cancerous) populations are heterogeneous and a fraction of resistant cells typically escape death. The subpopulation of malignant cells with protective mechanisms eventually replaces the original populations of susceptible cells.
The first of the single agents, folic acid antagonists, targeted DNA biosynthesis. Se V T DeVita, New Engl. J. Med. 298:907-910 (1978). Both replication of DNA and cell division, associated with the S phase, have been targets of chemotherapy. Examples of TCI targeting DNA synthesis have included antimetabolites, alkylating agents, natural toxins or antibiotics, platinum coordination complexes, and substituted urea. Known actions of these agents have been discussed by Calabresi, supra.
An agent that can act as a TCI can initiate cell damage during the cell cycle hierarchy in various ways. For example, a TCI can inhibit enzymes, compete for substrates, inhibit the transcriptional, translational or post-translational steps in molecular biosynthesis, introduce transcriptional or translational errors, disrupt molecular conformational changes, inhibit molecular transport, compete for energy transfer molecules, interfere with macromolecular polymerization, form molecular crosslinks, alkylate or cause strand breaks in DNA, or intercalate into the DNA helix. Thus, a TCI may impair cell cycle processes such as RNA transcription and translation, DNA strand elongation, replication, repair, supramolecular organization or separation, molecular transport. or macromolecular segregation. Alternatively, it may selectively injure any of the multiple cellular organelles associated with successful completion of specific subsets of the cell cycle hierarchy.
Another possible mode of selective damage by a TCI in neoplastic cells is a loss or deficiency of a checkpoint control, such as a cyclin-dependent kinase (cdk or cdc), which normally controls the cell cycle hierarchy. The role of checkpoint controls has been defined by observed effects of agents or mutations which relieve order dependence. See H A Crissman et al., Proc. Natl. Acad. Sci. (USA) 88:7580-84 (1991); Kastan, supra.; Murray, supra. (1992); Hartwell, supra. The cell cycle of normally cycling cells must traverse the G1 decision point (START) which commits a cell to continue through S phase resulting in DNA replication. Heichman, supra. Thus, cells exposed to a TCI prior to START may be partially protected from DNA damage by a delay within G1. This G1 delay can be mediated by the tumor suppressor p53 and enables cells to repair damaged strands of DNA prior to replication. Kastan, supra. Damage to DNA after START or DNA damage and bypass of START can be biologically deleterious, P M O""Connor et al., Cancer Res. 4776 (1994), possibly leading to DNA replication infidelity in S phase, with resulting genetic instability and ultimately premature cell death. Hartwell, supra; Kuerbitz, supra.; Shaw et al., Proc. Natl. Acad. Sci. USA 89:4496-9 (1992); T. Weinert, Semin. Cancer Biol. 4:129-140 (1993).
The actions of agents targeting S phase in eukaryotic cells are intrinsically complex due to the nature of DNA replication in S phase. For example, replication origins are discontinuous, chain elongation proceeds asynchronously, and progression at replication forks may be irregular. C S Newlon, Science 262:1830-31 (1993); V Levenson et al., Nucleic Acids Res. 21:3997-4004 (1993). Even as the DNA strands replicate in parallel process hierarchies within the overall cell cycle hierarchy, however, they share critical enzymes or metabolic intermediates. Murray, supra (1992); Laskey et al., Science 246:609-613 (1989); Nurse, supra; Heichman, supra.
Modern approaches to cancer chemotherapy developed during a time when knowledge of the cell cycle was advancing rapidly. Thus, it was recognized that neoplastic cells are vulnerable to agents that act during the S phase of the cell cycle. To better study the S phase, anti-metabolic agents were used to inhibit enzymes associated with purine or pyrimidine nucleotide biosynthesis affecting the ability of the DNA to replicate. These included ribonucleotide reductase (RNR) inhibitors, such as dThd or hydroxyurea (HU); dihydrofolate reductase inhibitors, such as methotrexate (MTX); or DNA polymerase inhibitors such as aphidicolin (Aph) to completely arrest progress of the target cells through the cell cycle at G1/S. G Galavazi et al., Exp. Cell Res. 41:428-51 (1966); D Thomas et al., Cell 5:57-32 (1975); T Ashihara et al., Methods Enzymol. 58:248-262 (1979); Levenson, supra.
It also was established that an excess of the normal metabolite dThd could reversibly arrest DNA replication in many cell lines of malignant origin or other proliferating cells. D. Kufe et al., Cancer Treat. Rep. 64:1307-1317 (1980).
In other studies, the use of excess dThd was found to be less damaging than MTX or HU and removal of dThd could be followed by a synchronous progression of cells through the remainder of the cell cycle. H R Zielke et al., Methods in Cell Biology 8:107-121 (1974); R E Meyn et al., Methods in Cell Biology 9: 103-113 (1975). As a result, repetitive synchronization of the cell cycle with dThd produced relatively pure cell populations in S phase. Zielke, supra.
Use of RNR inhibitors such as dThd or HU as a single agent in high dosages was also explored in cancer therapy. Many of the published reports concerning use of dThd have been critically reviewed. See Ellims, supra; O""Dwyer et al., Cancer Res 47:3911 (1987); S O Ooi et al., Experientia 49:576-81 (1993). Some successful results in patients with leukemia, lymphomas, or solid tumors were reported. D W Kufe et al., Cancer 48:1513-6 (1981); A Levya et al., J Cancer Res. Clin. Oncol. 107:211-216 (1984); R L Schilsky et al., Cancer Res. 46:4184-4188 (1986). Blood levels of up to 6 mM dThd could be achieved with oral doses. M S Blumenreich et al., Cancer Res. 44:2203 (1984); O""Dwyer, supra. In general, however, the use of dThd as a single agent chemotherapy was considered marginally potent for damage to malignant cells. Toxic side-effects often were intolerable at the dosages required to produce any therapeutic benefits.
B. Combination Chemotherapy
In cancer therapy, the survival of even a few malignant cells is more critical than in anti-infective therapies, since host immune mechanisms for killing of malignant cells typically are not effective. Therefore, exogenous cell killing plays a major role in prolonging clinical remissions or achieving cure. In the context of conventional chemotherapy, however, cell killing is described by first order kinetics: increasing doses of a single TCI will selectively damage an increasing percentage of the remaining malignant cells, but cannot destroy every potentially malignant cell without sacrificing the host. Calibresi, supra. Mathematically, this is analogous to Zeno""s paradox of fast and slow runners (W L McLaughlin, Sci. Amer. 271:84-89, 1994).
Since the 1960s, heavy reliance has been placed upon combinations of agents to produce more durable clinical responses than are possible with single agents. R L Capizzi et al., Sem. Oncol. 4:227-253 (1977); DeVita et al., Cancer 35:98-110 (1975). DeVita, supra (1994), discusses the generally agreed objectives of agent combinations: to maximize cell killing with tolerable toxicity, to provide coverage of cancer cells with differing levels of vulnerability in a heterogeneous tumor population, and to prevent or slow the evolution of neoplastic clones that develop increasing resistance.
DeVita also sets forth several principles in the current selection of agent combinations:
(i) each agent should be effective as a single agent in cell killing;
(ii) agents should be combined from different classes of actions to allow maximum dose intensity;
(iii) additive patient morbidity or mortality should be avoided; and
(iv) schedules or intervals of agent administration should be optimized.
As mentioned above, development of cell resistance to cytotoxic agents may involve mutations in p53 or other cell cycle control genes and may be accompanied by abnormalities in the cell cycle order dependence or checkpoint controls. C S Morrow et al., Ann. NY Acad. Sci. 698:289-312 (1993). Appropriate selection of multiple agents and achievement of high dose intensity are currently perceived as the critical issues in the design of chemotherapeutic protocols to avoid the development of that resistance.
Efforts have been aimed at modulating the cell cycle as a means for increasing cell damage by combinations of chemotherapy agents. The objective was to maintain malignant cells within the S phase of the cell cycle, where they may be most vulnerable to damage. See H O Klein et al., Semin. Hematol. 11:203-27 (1974). R L Stolfi et al., Pharmac. Ther. 49:43-54 (1991), have referred to these strategies as xe2x80x9ccytokinetic modulationxe2x80x9d.
Some uses of MTX, dThd, or pyrimidine analogs to arrest cell populations at a specific point in the cell cycle to modulate synergistic killing by application of a successive cytotoxic agent have been tested. B Bhutan et al., Cancer Res. 33:888-894 (1973); Ellims, supra; S D Henderson et al., Invest. New Drugs 5:142-154 (1987); Stolfi, supra. This approach often has been referred to as xe2x80x9csynchronizationxe2x80x9d of the cell cycle. Capizzi, supra. Cells exposed to high concentrations of anti-metabolites are detained within a limited subset of the cell-division cycle hierarchy, typically at a specific point within late G1 or early S phase. Thus, few or no cells in the population can proceed beyond this point of detention. Therefore, this type of effect is better described either as a cell cycle xe2x80x9carrestxe2x80x9d or a xe2x80x9cstatic synchronizationxe2x80x9d. W Vogel et al., Hum. Genet. 45:193-8 (1978).
A number of other efforts to control cancer cell growth by manipulating the cell-division cycle have been directed to altering the cell cycle distribution within the cell population targeted for damage. Other protocols were designed to stimulate malignant cells from G0 phase or G1 phase into proliferative status and thus increase their vulnerability to anti-metabolic drugs acting during DNA replication. H H Euler et al., Ann. Med. Interne. (Paris) 145:296-302 (1994); B C Lampkin et al., J. Clin. Invest. 50:2204-14 (1971); Alama et al., Anticancer Res. 10:853-8 (1990). Conversely, other protocols were designed to prohibit normal cells from entering S phase and thus protect them from unintended damage by anti-metabolites. Capizzi, supra.
Agents in combination may have additive, synergistic or antagonistic effects. Intuitively, it might be supposed that a combination of agents causing a cell cycle arrest or static synchronization of the malignant cells would circumvent the problem of first order kinetics, combined dosages can be increased to a level sufficient to fill all malignant cells without sacrificing the host. Restriction of a malignant cell population to a limited set of the cell cycle hierarchy, where the cells were specifically vulnerable to damage by a successive TCI, might be expected to shift the dynamics of cell killing toward greater efficiency, and reduce side-effects by diminishing the cumulative time of host exposure to a TCI.
However, in actual trials, strategies of cell cycle arrest or static synchronization often have been disappointing. Capizzi, supra. This is due to an essential incongruity of the procedure. When the cell cycle is arrested, all cells are within a specific fraction of the cell cycle. If this subset of the cell cycle does not completely overlap the subset of the cell cycle where the TCI is most effective, the cell cycle arrest will not result in synergistic or even an additive action of the successive TCI. Cell cycle arrest or static synchronization can be advantageous only when the affected phase of the cell cycle actually encompasses a relevant target interval of the successive TCI. The target interval of a successive TCI may be located downstream and not within the kinetic boundaries of the arrested or statically synchronized population. Since a high concentration of an agent effecting cell cycle arrest or static synchronization can also act as a single agent TCI, some cell killing or damage will occur even prior to the addition of a successive TCI. Indeed, if a successive TCI were actually the more potent killing agent and acted downstream from the point of cell cycle arrest, then cell cycle arrest or static synchronization would actually protect cells from the successive agent. This problem was exemplified in previous trials with dThd used in high doses as a synchronizing agent. Doses of dThd required to achieve effective blood concentrations for cell cycle arrest ( greater than 3 mM) often caused toxic side effects which were not well tolerated by patients, and could not be justified in relation to the therapeutic effect. In one set of trials, the stated objective was to deliver maximally tolerable doses, and blood levels of up to 6 mM were achieved with oral doses. Blumenreich, supra. O""Dwyer, supra. This concentration range had been used successfully in vitro to prohibit cells in G1-phase from entering S phase (i.e. a G1/S block). J H Kim Biochem Pharmacol 14:1821-9 (1965); Littlefield, supra.; Kufe, supra, but it did not prove sufficiently potent to induce clinical inhibition of malignant cell growth in the patients. Thus, secondary toxicities proved difficult for patients to tolerate and the further use of dThd was discouraged and its use is no longer advocated or reported. Blumenreich, supra; O""Dwyer, supra; Ooi, supra.
Most of the agents previously used in chemotherapy to effect cell cycle arrest or static synchronization in chemotherapy have probably acted late in G. phase or early in S phase. Capizzi, supra. Therefore, they would be unlikely to potentiate TCI with actions beginning later in the cell cycle hierarchy, e.g., later in S phase.
In modified approaches to static synchronization for cancer chemotherapy, a number of investigators noted that the scheduling of particular drug combinations was critical for production of synergistic lethal effects either in vitro or in vivo. Capizzi, supra; Stolfi, supra. However, many of these combinations have only been tested on a trial and error basis without a clear rationale for the agent sequence, concentration ratios, schedule or duration employed. See A L Adel et al., Cancer Invest 11:15-24 (1993). In clinical parlance, therapeutic approaches of combining cell cycle arrest with sequential applications of a second agent have been referred to as xe2x80x9cschedule dependent enhancement,xe2x80x9d e.g., Capizzi, supra., or xe2x80x9cbiochemical modulation.xe2x80x9dF M Muggia et al., Semin. Oncol. (3 Suppl) 9:90-3 (1992).
One special strategy of cell cycle manipulation was referred to as xe2x80x9cpulse dose chemotherapyxe2x80x9d. R E Moran et al., Cancer Treat. Rep. 64:81-6 (1980). In this particular approach, leukemic tumor cells in mice were detained in S phase of the cell cycle during infusion treatment of the mice with hydroxyurea (HU). After the infusion of HU was ended, the cells were xe2x80x9creleasedxe2x80x9d to continue transit of the cell cycle. At finite times after termination of the HU infusion, experimental animals were treated with a xe2x80x9cpulsexe2x80x9d of a second agent (Ara-C). This method can be compared to the arrest method of H R Zielke, supra and J L Littlefield et al., 1974, in which an arrest of the cell-division cycle at a specific detention point is reversed so that the cells then move in concert through the cell cycle at a normal or possibly an accelerated rate.
The intention of xe2x80x9cpulse dose chemotherapyxe2x80x9d was to maximize impact of the second agent as cells were moving in concert through the cell cycle. Mean survival time of the mice was determined. Mice treated with Ara-C at zero time, just after the HU infusion ended, showed improved survival, but treatments with Ara-C at later times after stopping the HU infusion did not potentiate the effect of HU. This procedure of cell cycle synchronization followed by a second agent relies on the nonsimultaneous action of the two agents. Moreover, the indirect results with respect to mean survival time of the animals cannot be directly translated to effects on tumor cell damage.
A major challenge in combination chemotherapy is to determine an optimal synergy in view of the multiple variables of dose, pharmacokinetics, sequence, and scheduling. Even for two agents, the most effective utilization is not necessarily clear from a simple combinational analysis of the optimum for each agent. Empirical variables include the dose or effective concentration of each agent, sequence of agents, intervals between doses, i.e., schedule, duration of dosages and numbers of doses per course of therapy. See Capizzi, supra; Adel, supra. See also M C Berenbaum, Pharmacol. Rev. 41:93-141 (1989).
In vitro testing using tissue cultures or testing in animal models can provide guidance on proposed combinations prior to clinical application. J Plowman et al., Cancer Res. 55:862-7 (1995); M E Wall et al., Cancer Res. 55:753-60 (1995); J Higashihara et al., Gynecologic Oncology 48:171-179 (1993); Berenbaum, supra. Rev. 41:93-141 (1989); P C Schroy III et al., Cancer Res. 48:3236-3244 (1988); R H Shoemaker et al., Cancer Res. 45:2145-53 (1985); Capizzi, supra.
Technical methods of in vitro evaluation of chemotherapeutic agents have varied, but, measuring inhibition of cell growth by staining or dye uptake is accepted by the National Cancer Institute as a method for quantitative analysis. Plowman, supra. Uptake of 3H-thymidine to obtain the labelling index is another common method of analyzing tumor cell sensitivity to chemotherapeutic agents. G H Baltuch et al., Neurosurgery 33:495-501 (1993); I P Hayward et al., Int. J. Cell Cloning 10:182-9 (1992); Schroy, supra. Differences in nucleic acid salvage pool sizes or thymidine kinase activities can make this approach unreliable. A similar approach is provided by the technique of flow cytometry in which antibodies against halogenated pyrimidine analog or a Hoechst dye track cells in S phase. Perez, supra; J P Perras et al., Cytometry 14:441-8 (1993); P Ubezio et al., Cytometry 12:119-126 (1991); M Poot et al., Biochem. Pharmacol. 41:1903-9 (1991).
Classically, the synergistic interaction of two agents is assessed using a series of dose-response curves from which fractional inhibitory concentrations can be calculated. G B Elion et al., J. Biol. Chem. 208:477-88 (1954); Berenbaum, supra; G M Eliopoulos et al., Chapter 13, pp. 432-492, IN: Antibiotics in Laboratory Medicine, 3d ed. V Lorian ed. The data can be obtained by a checkerboard technique, see Lorian, supra; Howard et al., Int. J. Cell Cloning 10:182-9 (1992). This method of analysis is known as an isobologram.
There are several practical problems with the isobologram method. First, the dose response curve of many agents used in chemotherapy becomes nonlinear at very high concentrations. Second, if agents to be compared prove asymmetrically potent or weak in single use, comparisons may be impossible. Third, results could be highly variable as coefficients of synergy calculated from an isobologram can be different for each fixed level of cytotoxicity.
Thus, collection of data for isobolograms can be laborious, and the isobologram method has limited scope for demonstrating an optimal range of agent combinations for practical effects. Berenbaum, supra. Efforts to resolve this shortcoming have proposed analyses of data as a three dimensional surface construct. W R Greco et al., Cancer Res. 50:5318-27 (1990) or other special methods (R C Li et al. Antimicrobial Agents and Chemotherapy 37:523-531, 1993. However, these solutions ultimately involve isobole data presentation. The problem becomes inordinately complex for therapeutic strategies involving multivariate interactions of more than two agents.
C. Other Cytotoxic Agents Affecting the Cell Cycle
Other recent efforts to relate the application of chemotherapeutic agents to cell cycle events have focused upon the role of checkpoint controls, Oxe2x80x94Connor, supra (1992), or the regulation of apoptosis by manipulation of the induction of p53 gene product or related products of p21Waf1/CIP1, W S El-Deiry et al., Cell 75:817-825 (1993).
For instance, protein kinases play an important role in neoplasia. Overexpression has been associated with hematologic malignancy. G Q Daley et al., Science 247:824-30 (1990). Neoplastic cells may be deficient in kinase-mediated control of progression through G1 and commitment to DNA replication. High levels of several protein kinases in cancer cells have been associated with multidrug resistance to conventional chemotherapeutic agents which are targeted to S phase. Baltuch, supra; J A Posada et al., Cancer Commun. 1:285-92 (1989); K Kawamura, Hokkaido Igaku Zasshi 69:354-71 (1994).
Use of protein kinase inhibitors in cancer control appears promising, since some are very potent toxins, but are less likely to be mutagenic than conventional agents which alkylate or crosslink DNA. C A O""Brian et al., J. Natl. Cancer Inst. 82: 1734-5 (1990); S Akinaga et al., Cancer Chemother. Pharmacol. 33:273-80 (1994); G K Schwartz et al., J. Natl. Cancer Inst. 85:402-7 (1993). The potent protein kinase inhibitor staurosporine (STSP) and several functional analogues have been of interest since they can (1) reverse or modulate multidrug resistance, K E Sampson et al., J Cell Biochem 52:384-95 (1993); C H Versantvoort et al., Br. J. Cancer 68:939046 (1993); K Miyamoto et al., Cancer Res. 53:1555-9 (1993); I Utz et al., Int. J. Cancer 57:104-10 (1994); (2) arrest cell cycle progression, S Bruno et al., Cancer Res. 51:470-473 (1992); Crissman, supra.; or (3) induce apoptosis, Bertrand et al., Exp. Cell Res. 211:314-321 (1994); D W Jarvis et al., Cancer Res. 54:1707-14 (1994).
STSP is a product of Streptomyces staurosporous, Meksuriyen D and Cordell G A, J of Nat. Products 51:893-899 (1988), and is one of the most powerful broad spectrum inhibitors of protein kinases, Tamaoki, Methods Enzym. 201:340-347 (1991). In addition to actions on protein kinase C and tyrosine kinases, C D Smith et al., Biochem. Biophys. Res. Comm. 156:1250-1256 (1988), STSP inhibits cyclin-dependent kinases associated with the S/G2 transition, D M Gadbois, Biochem. Biophys. Res. Comm. 189:80-85 (1993), and it can arrest neoplastic cells in G2 phase of the cell cycle.
Work within human glioma cell lines, see Baltuch, supra, and an appended editorial comment by P L Komblith, as well as previous reports by Schwartz, supra, and studies of its effects on multidrug resistance, discussed herein, indicated that STSP has potential in cancer chemotherapy. However, there have been no reports to date of clinical trials in humans. The work in rats and dogs by R A Buchholz et al., In Cellular and Molecular Mechanisms in Hypertension, p. 199-204, Plenum Press, NY (1992) and Hypertension 17:91-100 (1991), suggest that human plasma levels of 500 nM might be testable (see Table). However, this is not yet certain.
The kinase inhibitor agents K252A, KT5720, and KT5926, have different ranges of potency with regard to inhibition of protein kinases. They are described in a series of references: W E Payne et al., J. Biol Chem. 263:7190 (1988); R L Raynor, J. Biol Chem. 266:2753 (1993); C. Schachtele et al., Biophys Biochem Res Comm 115:542 (1968); H Kase et al., Biochem Biophys Res Comm 142:436 (1987); S Nakanishi et al., Mol Pharmacol 37:482 (1990); W H Fletcher et al., J Biol Chem 261:5504 (1986); and H C Chang et al., J Biol Chem 261:989 (1986). They have not been tested clinically as yet.
D. Problems with Chemotherapy
Several problems are widely recognized in the current use of TCI for chemotherapy and the other purposes. The first problem is non-specificity. A TCI may not be sufficiently selective, resulting in the injury of cells not intended for damage. See Id., E M Ross, Chapter 2, p. 33-48, Pharmacodynamics: Mechanisms of drug action and the relationship between drug concentration and effect, IN: Goodman and Gilman""s The Pharmacologic Basis of Therapeutics, 8th ed., (A G Gilman et al., ed., Pergammon Press, New York, 1990). A second problem is heterogeneous vulnerability where the inherent genetic variability of cells in a population intended for killing can frustrate efforts to achieve absolute and specific lethality. S Calabresi, supra. A third problem is acquired resistance, where some fraction of the cells intended for damage by a TCI acquire resistance to the TCI by physiological or metabolic adaptation, or by genetic mutation. Id.
In single or multiple clinical chemotherapies, non-specific xe2x80x9cside-effectsxe2x80x9d may become noxious and intolerable, resulting in significant patient morbidity. S M Pirisi et al., New Engl. J. Med.330:1279 (1994); S M Grunberg et al., New Engl. J. Med. 329:1790-1796 (1993); O""Dwyer, supra. In clinical pharmacotherapeutics, it is considered beneficial to increase the selectivity or xe2x80x9ctherapeutic indexxe2x80x9d of a cytotoxic agent. Gilman, supra. Thus, important objectives of therapeutic drug development or improvements in therapeutic drug application include efforts to increase the ratio of specific cytotoxic benefits, e.g., the intended killing or damage of a designated cell population, to non-specific side effects which produce host morbidity or environmental disruptions. In addition, there is also a need to develop more potent pharmaceuticals.
A major limitation of cancer chemotherapy has been perceived to be the inability to escalate doses of effective anticancer agents, such as TCIs, into the high end of dose-response curves due to intolerable side effects. DeVita, supra (1994). It is also a current concern that omission of one agent from a designed combination may allow overgrowth by a cell lineage susceptible to that agent, but resistant to other agents. Another concern is that the use of an effective agent in less than maximum strength may vitiate the objectives of a combined agent protocol.
An increase in therapeutic TCI effects would be valuable during both primary induction or adjuvant chemotherapy, R Arriagada et al., New Engl. J. Med. 329:1848-52 (1993); W C Wood et al., New Engl. J. Med. 33:1253-9 (1994); for post-remission chemotherapy, R J Mayer et al., New Engl. J. Med. 331:896-903 (1994); for high dose chemotherapy followed by autologous hematopoietic rescue, W P Peters, et al., J. Clin. Oncol. 11:1132-43 (1993); A M Marmont, Lupus 2:151-6 (1993); for extracorporeal purging of malignant cells from tissues intended for transplantation, F. Sieber and M. Sieber Blum 46:2072-6 (1986); F. Lin et al., Cancer Res. 52:5282-90 (1992); or for debulking of metastatic tumor in body cavities, M E L van der Burg et al, New Engl. J. Med. 332:629-34 (1995); R. Arnold, Eur. J. Clin. Invest. 20 Suppl 1:S82-S90 (1990).
Therefore, there is a need in the art for new combinations of cell killing agents, including new combinations of dosage strategies, whereby the first agent modulates the cell cycle so as to maximize the toxic effect of the second agent on target cells, while minimizing the toxic effect on non-target cells. This need in the art is particularly acute in the area of cancer chemotherapy.